Alveolar epithelium is comprised of two distinct cell populations, type II (AT2) and type I (AT1) cells. During repair of lung injury, AT2 cells proliferate and subsequently differentiate into AT1 cells in order to restore the alveolar epithelial barrier. Transcriptional programs that mediate transitions between AT2 and AT1 cell phenotypes, and that regulate differentiation within adult alveolar epithelium, are poorly understood. In the context of lung regeneration, acquisition of knowledge about specific TF essential for AT1 cell commitment is a critical step toward reprogramming AT2 cells to direct differentiation to AT1 cells in order to augment normal repair. We have developed an in vitro culture model in which alveolar epithelial cell (AEC) differentiation can be experimentally modulated, providing a useful tool with which to investigate transcriptional pathways that regulate AEC differentiation. We identified FoxO1, a member of the FoxO subclass of forkhead transcription factors (TF), that is upregulated in AEC that have transdifferentiated toward an AT1 cell-like phenotype on later days in culture. FoxO TF regulate diverse cellular functions, including cell cycle and differentiation. Their precise functions are context-dependent and modulated by post-translational modifications and interactions with other TF. The goal of this proposal is to investigate the role of forkhead TF, and specifically FoxO1, in programming AEC transdifferentiation from AT2 to AT1 cell phenotype in order to elucidate signaling-coupled transcriptional networks important for maintenance and repair of alveolar epithelium. We hypothesize that FoxO1 plays an essential role in AT1 cell commitment, differentiation and survival in adult lung, regulating transitions from AT2 to AT1 cell phenotype and promoting quiescence following injury and/or exposure to growth factors. We will use our well-characterized model of AEC differentiation in vitro, in conjunction with freshly isolated AT2 and AT1 cells, our expertise in characterizing AEC differentiation, and genetically modified mice with AT2 or AT1 cell-specific deletion of FoxO1 to address the following Specific Aims: 1) characterize FoxO1 expression and activity in AEC; 2) evaluate molecular interactions between FoxO1 and AEC-specific TF; 3) explore the role of FoxO1 in regulation of AEC transdifferentiation; and 4) investigate the role of FoxO1 in alveolar epithelial repair following injury in mice with epithelial-specific deletion of FoxO1. We will evaluate FoxO1 expression/activity and regulation as a function of AEC phenotype, characterize interactions between FoxO1 and other TF in regulating AEC- specific genes, assess the role of FoxO1-associated epigenetic modifications in modulation of AEC-specific gene expression, and investigate effects of deletion of FoxO1 in vitro and in vivo on AEC differentiation and repair following injury. Characterization of essential TF required to induce AT1 cell differentiation is a critical step toward understanding alveolar epithelial regeneration following injury. PUBLIC HEALTH RELEVANCE: Failure of type II (AT2) cells to divide and give rise to AT1 cells to restore the epithelial lining following injury can lead to lung scarring and death from respiratory failure. The mechanisms that direct AT2 cells to change into AT1 cells are poorly understood. Characterization of regulatory (transcription) factors required to induce AT2 cells to give rise to AT1 cells is a critical step toward understanding alveolar epithelial regeneration following injury and identifying potential therapeutic targets to enhance lung repair.